Sialic acids are acyl derivatives of neuraminic acid. N-Acetylneuraminic acid [FIG. 1] is one of the most frequently found sialic acids. (Blix, G., Gottschalk, A., and Klenk, E. (1959) Nature 179:1088). Sialic acids have been the subject of a great deal of research because they play several important and intriguing biological roles. For example, sialic acids play a role in cell biology by their negative charge, by influencing the conformation of glycoproteins, by acting as receptors for microorganisms, toxins and hormones and by masking receptors and recognition sites of molecules and cells. (Schauer, R. (1987) "Analysis of Sialic Acids" Methods in Enzymology 138:132-161).
Through research on sialic acids, a large number of sialidases have been isolated. In general, sialidases are a class of enzymes capable of cleaving the glycosidic linkage of sialic acid. [FIG. 2]. To date, all sialidases that have been isolated release free sialic acid as the enzymatic product. (Schauer, R. (1982) "Chemistry, Metabolism and Biological Functions of Sialic Acids" Advances in Carbohydrate Chemistry and Biochemistry 40:131-234).
2,7-Anhydro-N-acetylneuraminic acid, a relative of sialic acid, has also been isolated. [FIG. 1]. Gross, S. K , et al., (1975) Carbohydrate Res. 41:344-350, reported detection of 5-acetimido-2,7-anhydro-3, 5-dideoxy-D-glycero-D-galacto-nonulopyranose in the acid hydrolysate of glycosidically-linked sialic acid in which the carboxylic function had been reduced to an alcohol. Lifely, M. R., et al., ((1982) Carbohydrate Res. 107:187-197), showed that methanolysis of sialic acid gave the methyl ester of 2,7-anhydro-N-acetylneuraminic acid in addition to the methyl ester ketoside of sialic acid. The presence of 2,7-anhydro-N-acetylneuraminic acid in rat urine was reported by Schroder, et al. (1983) "Proc 7th Int. Symp. Glycoconjugates", Chester, Mass., eds., Rahms, Lund, Sweden, pp. 162-163. 2,7-Anhydro-N-acetyneuraminic acid has also been detected in human wet cerumen (ear wax) by Suzuki, M., et al., (1985) J. Biochem (Tokyo) 97:509-515.
The presence of 2,7-anhydro-N-acetylneuraminic acid in human wet cerumen (ear wax) suggests that this unusual sialic acid derivative may have bactericidal activity. Since 2,7-anhydro-N-acetylneuraminic acid is resistant to degradation by sialyl-aldolase, this compound can also serve as a reservoir for sialic acids in the biological system. The biological function of 2,7-anhydro-N-acetylneuraminic acid is still largely unknown. This is due to the lack of an effective synthetic method for its production.
The close relationship of 2,7-anhydro-N-acetylneuraminic acid to sialic acid, both having a stereochemically identical carbon backbone, is evident when one compares the Fischer diagram representations of N-acetylneuraminic acid and 2,7-anhydro-N-acetylneuraminic acid. [See FIG. 1(a)]. However, 2,7-anhydro-N-acetylneuraminic acid has a three-dimensional structure that is distinct from sialic acid. [FIG. 1(b)]- Of significance is the bicyclic ring structure of 2,7-anhydro-N-acetylneuraminic acid. Unlike sialic acid which is in a .sup.2 C.sub.5 conformation with all of its pyranose ring substituents equatorial except the substituents at the anomeric carbon (C-2), 2,7-anhydro-N-acetylneuraminic acid is in a .sup.5 C.sub.2 conformation with all of the pyranose ring substituents axial. Furthermore, N-acetyl-neuraminic acid is a reducing sugar while 2,7-anhydroneuraminic acid is non-reducing. Thus, despite the similarity of sialic acid and 2,7-anhydro-N-acetylneuraminic acid based on their Fischer representations, the three-dimensional structures and chemical properties of these two compounds are quite distinct. Hence, sialic acid and 2,7-anhydro-N-acetylneuraminic acid are structurally distinct and dissimilar compounds.
Recently, Y. T. Li reported the presence of an enzyme, named "Sialidase-L", in a leech that exhibits novel specificity and produces 2,7-anhydro-N-acetylneuraminic acid as the unexpected cleavage product. In addition to the synthetic substrate, 4-methylumbelliferyl-N-acetylneuraminic acid, sialidase-L was reported to selectively cleave sialic acid linked .alpha.2.fwdarw.3 to D-galactose from substrates including fetuin, .alpha.1-acid glycoprotein, neuraminlactose, and whale nasal keratan sulfate to yield 2,7-anhydro-N-acetylneuraminic acid as the sole observed product. [FIG. 3]. Sialidase-L does not cleave sialic acids that are linked .alpha.2.fwdarw.6 to D-galactose, .alpha.2.fwdarw.6 to N-acetyl-D-galactosamine or sialic acid linked .alpha.2.fwdarw.8 or .alpha.2.fwdarw.9 to N-acetylneuraminic acid. Furthermore, sialidase-L does not convert free sialic acid to 2,7-anhydro-N-acetylneuraminic acid. See Abstract for the 21st annual meeting of the Society for Complex Carbohydrates, Nashville, Tenn., Nov. 11-14, 1992, Glycobiology. volume 2, p. 459, 1992.
No specific information regarding the procedure for the isolation and properties of sialidase-L were disclosed at the meeting of the Society for Complex Carbohydrates. Isolation of sialidase-L has significant utility since it enables the development of methodology for producing 2,7-anhydro-neuraminic acid. Furthermore, sialidase-L enables selective cleavage of sialic acid-.alpha.2.fwdarw.3D-galactose glycosidic linkages in sialoglycoconjugates.